Method for influencing kinase pathways with IL-22

ABSTRACT

Interleukin-22 interacts with its receptor, referred to as IL-22R, and instigates a series of reactions, leading to activation of various molecules, such as JAK-1, Tyk2, and others. One can identify molecules which mediate this interaction by measuring the activity of one or more of the molecules in the pathway, to identify agonists and antagonists. These, in turn, are useful therapeutic agents, where inappropriate expression of one of the activated molecules is at issue, and requires amelioration.

RELATED APPLICATIONS

[0001] This application is a continuation in part of application in Ser.No. 09/915,735, filed Jul. 26, 2001, as well as a continuation in partof Ser. No. 09/626,617, filed Jul. 27, 2000, and Ser. No. 09/751,797filed Dec. 29, 2000, all of which are incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to various aspects of activation pathwaysinvolving the molecule referred to as interleukin-22, or “IL-22”. Inparticular, it relates to methods for mediating the interaction of IL-22and its receptors, as well as to methods for determining if IL-22expression is occurring, has occurred, and if so, at what levels.

BACKGROUND AND PRIOR ART

[0003] Interleukin-22, or “IL-22” hereafter, is an IL-10 relatedcytokine, that had previously been referred to as “TIF” or “IL-TIF” for“interleukin-10 related, T cell inducible factor.” See U.S. Pat. Nos.6,359,117; 6,331,163 and 6,274,710, as well as Dumoutier, et al., J.Immunol 164:1814-1819 (2000), all of which are incorporated by referencein their entirety. The molecule belongs to a family of cytokines withlimited homology to IL-10, including IL-10, IL-22, mda-7/IL-24, IL-19,IL-20 and AK155/IL-26. See Moore, et al., Annu. Rev. Immunol 19:683-765(2001); Dumoutier, et al., Eur. Cytokine Netw 13(1):5-15 (2002). Thecytokine shows 22% amino acid identity with IL-10. Functionally, IL-22activities which have been identified include upregulation ofacute-phase reactants in liver and hepatoma cells (Dumoutier, et al.,supra,) as well as induction of pancreatitis-associated protein (PAP 1),in pancreatic acinar cells (Aggarwal, et al., J. Interferon CytokineRes. 21:1047-1053 (2001)), suggesting a role for the cytokine ininflammatory processes. In addition, IL-22 has been shown to induce STATactivation in several cell lines, including mesangial cells, lung andintestinal epithelial cells, melanomas, and hepatomas. See Dumoutier, etal., supra; Dumoutier, et al., Proc. Natl. Acad. Sci USA 97:10144-10149(2000); also see patent application Ser. No. 09/626,617, filed Jul. 27,2000, incorporated by reference which referred to “TIF” as IL-21;however, the molecule has been renamed as IL-22.

[0004] The IL-22 molecule binds at cell surfaces to a receptor complexcomposed of two chains, which belong to the Class II cytokine receptorfamily, i.e., IL-22R and IL-10Rβ. See, e.g., Dumoutier, et al., Proc.Natl. Acad. Sci USA 97:10144-10149 (2000); Xie, et al., J. Biol. Chem275:31335-31339 (2000); Kotenko, et al., J. Biol. Chem. 276:2725-2732(2000); also see U.S. patent application Ser. No. 09/915,735, filed Jul.26, 2001, and incorporated by reference. This family of receptorsincludes receptors for type I and type II interferons, such as IFNAR1,IFNAR2, IFNGR1 and IFNGR2; IL-10Rα, IL-22R/CRF2-9, IL-10Rβ/CRF2-4,IL-20Rα/CRF2-8, IL-20Rβ/CRF2-11, and tissue factor. See Kotenko, et al.,Oncogene 19:2557-2565 (2000); Blumberg, et al., Cell 104:9-19 (2001);Kotenko, Cytokine Growth Factor Rev 217:1-18 (2002), all of which areincorporated by reference.

[0005] With the exception of IL-10R per se, signaling through thereceptors for IL-10 related, cytokines has not been investigated verywell. The binding of IL-10 to its receptor complex (IL-10Rα andIL-10Rβ), induces activation of JAK-1, and Tyk-2 tyrosine kinases.Experiments by Finbloom et al., J. Immunol. 153:1079-1090(1995), showedthat JAK-1 associates with IL-10Rα, and Tyk-2 copreciptates withIL-10Rβ. See Kotenko, et al., EMBOJ 16:5894-5903 (1997), regardingTyk-2. Activation of the two kinases, in turn, leads to phosphorylationof STAT1, 3 and 5. See Finbloom, et al., J. Immunol 153:1079-1080(1995); Wehinger, et al., FEBS Lett 394:365-370 (1996). In addition,IL-10 is known to activate PI3 kinase, and p70S6 kinase (Crawley, etal., J. Biol. Chem 271:16357-16362 (1996)), but not the MAP kinasepathway. Indeed, Sato, et al., J. Immunol 162:3865-3872 (1999), andGeng, et al., Proc. Natl. Acad Sci USA 91:8602-8606 (1994), show thatIL-10 inhibits this pathway in monocytes and dendritic cells.

[0006] The inventors have investigated the mechanism of action involvedin the binding of IL-22 to its receptor, and have discovered a pathwayof activation not reported previously. Since IL-22 and IL-10 share onereceptor subunit, i.e., IL-10Rβ, and the functional receptor complexinvolves IL-22R for IL-22, and IL-10Rα for IL-10, it was believed,initially, that the signalling pathways would be nearly identical;however, this has proven to not be the case. It has now been shown thatIL-22 induces phosphorylation of JAK-1 and Tyk-2, but not JAK-2. It hasalso been shown that JAK-1 is absolutely required for IL-22 signaling tooccur. While IL-10 also activates JAK-1 and Tyk-2, and both inducephosphorylation of the same STATs, it has now been shown that IL-22induces activation of ERK, JNK and p38 pathways, which IL-10 does not.In addition, it has been shown that IL-22 induces serine phosphorylationof STAT3, which IL-10 does not.

[0007] These, and other features of the invention will be evidenced inthe disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0008] It has been established previously by, e.g., Dumoutier, et al.,J. Immunol 164:1814-1819 (2000); Dumoutier, et al., Proc. Natl. Acad.Sci USA 97:10144-10149 (2000); Xie, et al., J. Biol. Chem275:31335-31339 (2000); Kotenko, et al., J. Biol. Chem 276:2725-2732(2000), that IL-22 induces phosphorylation of STAT1, STAT3 and STAT5.Experiments were designed to study the kinetics of phosphorylationfurther. Samples of rat hepatoma cell line H4IIE were grown inIscove-Dulbecco's medium, supplemented with 10% fetal calf serum, 0.55mM L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine. Otherhepatoma cell lines are known to express the IL-22R/IL-20Rβ complex, soit was presumed that H4IIE did so as well. These cells were thenstimulated with recombinant murine IL-22. The recombinant murine IL-22was produced by transiently transfecting HEK 293-EBNA human embryonickidney cells using the well known lipofectamine method. In contrast toH4IIE, cell line HEK 293 only expresses IL-10Rβ. Supernatants from thetransfected HEK 293-EBNA cells (1%), were combined with 4×10⁵ H4IIEcells, for 5, 15, or 30 minutes, or with control supernatant, for 15minutes. Cells were lysed, in 500 μl of Laemmli buffer, and boiled for 3minutes before loading on pre-cast, SDS-PAGE gels, and transferred,electrophoretically, to nitrocellulose membranes. The membranes wereblocked in 5% non-fat dry milk, washed, and then probed with antibodiesspecific for phosphorylated STAT1, STAT3 and STAT5. The membranes werethen reprobed with an anti actin-β antibody.

[0009] The results showed that phosphorylation of all three STATmolecules was induced within 5 minutes. Phosphorylation was transientfor STAT1 and STAT5, with levels decreasing to barely detectable after30 minutes. The phosphorylation of STAT3, however, could still bedetected at least an hour after stimulation.

[0010] These experiments were repeated using recombinant murine IL-22produced in E. coli in accordance with Dumoutier, et al., J. Immunol166:7090-7095 (2001), incorporated by reference. Similar results wereobtained.

EXAMPLE 2

[0011] These experiments confirmed that IL-22 induced STATphosphorylation correlated with transcriptional activation.

[0012] A total of 1.2×10⁷ H4IIE cells were electroporated, at 250V,200Ω, 1200 μF, with 50 μg, pGRR5-luc, or 30 μg pSRE-luc. The pGRR5-lucconstruct contains 5 copies of the STAT-binding site of the FcγRI gene,inserted upstream from a luciferase gene controlled by the TK promoter.The pSRE-luc construct contains repeats of the serum responsive elementof the c-fos promoter. In addition to the constructs described, supra,cells were transfected with 5 μg of reporter plasmid pRL-TK, as aninternal control.

[0013] Cells were seeded in 12 well plates at 10⁶/ml. The day afterplating, cells were stimulated for 3 hours with 2000 U/ml IL-22, or withcontrol medium prior to lysis. Luciferase assays were carried out usingcommercially available products.

[0014] The results indicated that when cells were electroporated withthe pGRR5-luc construct, IL-22 stimulation induced a 35 fold increase inluciferase activity.

EXAMPLE 3

[0015] It is well know that the JAK kinases are responsible for STATphosphorylation in response to cytokines. As such, experiments weredesigned to determine if JAK kinases are activated by IL-22, and if so,which of these is or are so activated.

[0016] To determine this, 5×10⁵ H4IIE cells were stimulated, asdescribed, supra, with IL-22 or control medium, and were eithersubjected to Western blotting, using an anti-phospho Tyk-2 antibody, orimmunoprecipitation using anti-JAK-1 or anti-JAK-2 antibodies.

[0017] With respect to Tyk-2, following Western Blot probing with theanti-phospho-Tyk-2 antibody, the membrane was re-probed, with anti-Tyk-2antibody.

[0018] With respect to the immunoprecipitation experiments, 3×10⁷ H4IIEcells were stimulated with the 1% HEK293 supernatant described supra,250 U/ml of gamma interferon which is known to activate JAK-2 and JAK-1,or control medium, for 5 minutes. These cells were then washed, andresuspended in 1 ml of lysis buffer (1% NP-40, 0.1% deoxycholate, 0.1%SDS, 50 mM Tris, pH8, 150 mM sodium chloride, 1 mM EDTA, 1 mM sodiumvanadate, 1 mM sodium fluoride, and inhibitor cocktail). Lysates werehomogenized by 5 passages through a 20 gauge needle, incubated for 45minutes on ice, and centrifuged (14,000×g). Following this, 2.5 kg ofanti-JAK-1 or anti-JAK-2 polyclonal antibody were added to thesupernatant, and incubated, overnight at 4° C. The lysates were thenincubated with protein A-agarose beads for two hours. The agarose beadswere washed, resuspended in Laemmli buffer (25 ml), and boiled. Proteinswere then separated on an 8% SDS-PAGE gel, transferred to anitrocellulose membrane, blocked in 1% bovine serum albumin solution,and were then incubated, overnight, with 1 μg/ml of antibodies specificfor phosphotyrosine. Any proteins were detected by chemiluminescence. Asa control, the membranes were reprobed with anti-JAK-1 or anti-JAK-2antibodies.

[0019] The results indicated that IL-22 stimulation of the H4IIE cellsinduced rapid phosphorylation of Tyk-2 and JAK-1, but not JAK-2.

[0020] Experiments were repeated using E. coli derived IL-22, andsimilar results were obtained.

EXAMPLE 4

[0021] These experiments outline further assessments of the functionalrole of JAK-1 in IL-22 signalling.

[0022] U4C is a fibrosarcoma cell line which is known to be JAK-1deficient. See Kohlhuber, et al., Mol. Cell. Biol 17:695-706 (1997).Samples of this cell line were transiently transfected with human IL-22RcDNA (500 ng),(Mizushima, et al, Nucleic Acids Res. 181:5322(1990)),pGRR5-luc (100 ng) and 100 ng of pRL-TK in accordance with Lejeune, etal., Biochem J. 353:109-116 (2001), incorporated by reference. Briefly,cells were seeded in 12 well plates at 4×10⁵ cells/well one day prior totransfection. Standard lipofectamine methods were used. Four hours aftertransfection, the cells were stimulated with one of: (i) control medium,(ii) human IL-22 (2000 U/ml).

[0023] Experiments were carried out in parallel, with cultures alsobeing transfected transiently with 40 ng of plasmid pRK5-JAK-1, whichencodes JAK-1, or empty vector.

[0024] The results indicated that IL-22 failed to induce luciferaseactivity in the transfectants, unless the cells were transfected withJAK-1 cDNA.

[0025] Additional experiments were carried out with parental cell line2C4, which does express JAK-1, and cell line γ2A, which is deficient inexpression of JAK-2. Both cell lines were able to, and did respond to,IL-22.

EXAMPLE 5

[0026] These experiments were designed to determine if IL-22 inducedphosphorylation of members of the MAPK pathway. To begin, 5×10⁵ H4IIEcells were seeded, in 6 well plates, one day before stimulation withrecombinant, murine IL-22 (2000 U/ml), for 10, 20, 30 or 40 minutes, orwith control medium for 40 minutes. In some experiments, cells werepreincubated for one hour with 50 μM of a known MEK1 inhibitor,“PD98059”, or 10 μM of U0126, which is also an MEK1 inhibitor. Followingincubation, cell lysates were analyzed, via Western blotting asdescribed supra, using an antiphospho-ERK1/2 antibody.

[0027] The results indicated that IL-22 induced sustainedphosphorylation of ERK1/2. The inhibitors, i.e., PD98059 and U0126, bothblocked phosphorylation completely. As these are both MEK inhibitors,this suggests that MEK activation is involved in phosphorylation ofERK1/2.

[0028] In follow-up experiments, Western blotting was carried out oncell lysates, in the manner described in this example, using antibodiesagainst phosphorylated forms of MEK1/2, p90RSK, JNK/SAPK and p38, all ofwhich are members of the MAPK pathway.

[0029] The results indicated that p90RSK was phosphorylated in responseto IL-22, which is in accordance with the results, since p90RSK is awell known substrate of ERK. IL-22 also induced delayed phosphorylationof JNK/SAPK and p38 MAP kinases.

[0030] Functional activation of the MAPK pathway was confirmed byadditional experiments paralleling those described supra. To elaborate,1.2×10⁷ H4IIE cells were electroporated with 30 μg of pSRE-luc, and 5 μgof pRL-TK vectors, as described supra. Cells were seeded in 12 wellplates following transfection, at 10⁶ cells/well. The day after seeding,cells were preincubed for 1 hour, in the presence of DMSO (1/1000 finaldilution), PD98059 (50 μM final), or U0126 (10 μM final), beforestimulation with murine IL-22 (2000 U/ml), or control medium. Luciferaseassays, as described supra, were carried out 3 hours after stimulation.

[0031] The data indicated that IL-22 stimulation induced a 2.25 foldincrease in luciferase activity, which was abolished completely whenthere was preincubation with any of the MEK inhibitors.

EXAMPLE 6

[0032] Follow up experiments were carried out to determine if relatedmolecule IL-10 functioned in the same way IL-22 did.

[0033] To test this, H4IIE cells were stably transfected with IL-10Rα.This was accomplished by subcloning murine IL-10Rα cDNA intopEF/Myc/Cyto plasmid, which carries a geneticin resistance gene. A totalof 1.2×10⁷ H4IIE cells were electroporated (20V, 200Ω, 1200 μF), with 50μg of IL-10Rα cDNA. The day after transfection, cells were cultured with2 mg/ml geneticin, until a bulk culture was secured.

[0034] Following stable transfection, 5×10⁵ transfected H4IIE cells wereseeded in 6 well plates. One date later, the cells were stimulated withIL-10 (10 μg/ml), or murine IL-22 (2000 U/ml) for 10, 20, 30 or 40minutes, or with control medium for 40 minutes. Total lysates wereanalyzed via Western blotting, with an anti-phospho-STAT3 antibody, andan anti-phospho-ERK1/2 antibody, followed by reprobing with ananti-actin-β antibody.

[0035] The IL-10 did not activate the ERK/MAPK pathway.

EXAMPLE 7

[0036] The preceding examples demonstrated that STAT3 was phosphorylatedin the mechanism described. STAT3 can be phosphorylated on tyrosine, butit can also be phosphorylated on a serine residue, in response tostimulation by cytokines such as IL-6. See Schuringa, et al., Biochem J347:89-96 (2000), incorporated by reference. To determine ifphosphorylation of serine was taking place, 5×10⁵ H4IIE cells wereseeded in 6 well plates one day before they were stimulated with murineIL-22 (2000 U/ml), for 10, 20 or 30 minutes, or with control medium for30 minutes. Experiments were run in parallel, both with and withoutpreincubation 1 hour prior to stimulation with 50 μM PD98059 or 10 μMU0126. Lysates were analyzed via Western blotting with an antibodyspecific for the serine phosphorylated form of STAT3. Reprobing of themembranes with an anti ERK1/2 antibody, and an anti-actin β antibody,followed.

[0037] Rapid serine phosphorylation was observed, with phosphorylationoccurring after only 10 minutes. While it has been reported that MAPKsmediate STAT Ser phosphorylation (Schuringa, et al., supra; Decker, etal., Oncogene 19:2628-2637 (2000); Lim, et al., J. Biol. Chem274:31055-31061 (1999)), when the H4IIE cells were preincubated with MEKinhibitors, the phosphorylation was slightly delayed, but not inhibited.

EXAMPLE 8

[0038] The experiments were designed to test the functional significanceof STAT3 serine phosphorylation. To test this, 1.2×10^(7 H)4IIE cellswere electroporated with 15 ug of pGRR5-luc, 5 ug of pRL-TK, and 15 ugof a vector which encoded wild type STAT3, or a mutated form, whereposition 727, normally a serine residue, was replaced by alanine whichprevents phosphorylation. See Schuringa, et al., FEBS Lett 495:71-76(2001). Five hours after transfection, cells were stimulated withcontrol medium, or murine IL-22 (2000 U/ml), for 3 hours. Luciferaseassays were carried out as described, supra.

[0039] The results indicated that the mutation at position 727 reducedluciferase induction from an 8-fold to a 4-fold increase upon IL-22stimulation, suggesting strongly that STAT3 serine phosphorylation isrequired for maximum activation.

[0040] To further investigate STAT3 serine phosphorylation, the effectof the STAT3 Ser727Ala mutant was tested on IL-10 inducedtransactivation. The IL-10 based experiments were carried out becauseIL-10 has never been described to phosphorylate STAT3 on a serineresidue. To test this, 4×10⁵ HEK 293 cells were seeded in 12 wellplates. The day after, these cells were transfected, either with avector encoding human IL-22R (500 ng) or one encoding murine IL-10Rα(500 ng), using the standard lipofectamine method. The cells were alsotransfected with a plasmid encoding the wild type STAT3 (1 ug) or theSer727Ala mutant form of STAT3 (1 ug) together with 100 ng pGRR5-luc,100 ng pRL-TK reporter plasmids and empty vector to give a total plasmidDNA content of 2 ug. Five hours after transfections, cells werestimulated with control medium, human IL-22 (2000 U/ml) or human IL-10(10 ng/ml) for 24 hours. Luciferase assays were carried out asdescribed, supra.

[0041] Simultaneously, cells were stimulated with IL-22, IL-10 orcontrol medium for 15 minutes before the lysis. The concentration of thestimulating agents was as described, supra.

[0042] Western blotting was carried out with total lysates, usingantibodies which detected either serine phosphorylated or tyrosinephosphorylated STAT3. Membranes were reprobed with anti-actin βantibodies.

[0043] The results indicated that IL-22 stimulation of the IL-22Rtransfected HEK293 cells resulted in a 6.5 fold increase in luciferaseactivity. Cotransfections with the STAT3 mutant reduced the activity to4 fold. In contrast, the cotransfection of the STAT3 mutant into cellsexpressing IL-10R had no effect on IL-10 induced transactivation.Further, Western blot analysis showed that IL-22, but not IL-10, inducedSTAT3 serine phosphorylation while both induced tyrosine phosphorylationof STAT3.

[0044] The preceding examples establish that the binding of IL-22 to areceptor leads to activation of STAT 1, 3 and 5, as well as theactivation of various kinases, including JAK-1, Tyk-2, MEK1/2,p90RSK,/SAPK and p38. Activation of STAT3 requires phosphorylation oftyrosine; however, when STAT3 is phosphorylated on tyrosine, it can befurther activated by serine phosphorylation. Hence, one embodiment ofthe invention is a method for screening to determine if a compound ofinterest mediates the effect of IL-22, especially the effect of IL-22 ona cell. This method involves, inter alia, combining the compound ofinterest with IL-22 or a portion of IL-22 sufficient to bind to an IL-22receptor, and a cell which expresses both an IL-22 receptor and at leastone molecule which is activated as a result of the interaction betweenIL-22 and its receptor, determining if said compound is activated, ifso, the degree of activation, and comparing the value obtained to acontrol value, wherein any difference there between indicates that thecompound of interest mediates the interaction between IL-22 and thereceptor. In this way, both antagonist and agonists can be determined.

[0045] The IL-22 used may be prepared recombinantly, which is preferred,or may have been prepared via purification from naturally occurringsources. The IL-22 may be IL-22 from any species which produces it,preferably mammal, such as murine IL-22, and most preferably, humanIL-22.

[0046] When produced recombinantly, the IL-22 may be produced viaexpression in prokaryotic cells, such as E. coli, or eukaryotic cells,such as the HEK293 cells, as is discussed supra; however, the skilledartisan will appreciate that other host cells may be used, as can cellfree transcription systems.

[0047] While the whole IL-22 molecule may be used, portions of an IL-22molecule which posses the ability to bind to an IL-22 receptor may beused. The determination of whether or not a portion of an IL-22 moleculecan be used in the assays of the invention can be determined veryeasily, such as by way of any of the assay mechanisms described in thepreceding examples, or any other method known to the skilled artisan.Also, variants of IL-22 which contain one or more amino acid additions,deletions, or substitutions, but which retain the ability to bind to areceptor may be used.

[0048] The determination of whether or not a interaction of IL-22 and areceptor compound is mediated can be measured in any number of ways. Forexample, it was shown, supra, that the induction of STAT phosphorylationcorrelates with transcription regulation. Hence, one way to determine ifa compound mediates the interaction of IL-22 and an IL-22 receptor is tomeasure the level of transcription of a substance that is mediated by aSTAT molecule, such as STAT1, STAT3, and/or STAT5. Such nucleic acidbased assays may be carried out using methods well known in the art. Forexample, one may use oligonucleotide probes specific to one or more ofthe genes whose transcription is mediated by one of the STAT molecules,such as STAT1, 3 and/or 5, and determine the amount of hybridization ofthe probes. The probes may be labelled, such as with a radiolabel, acolorimetric label, a biotin molecule, or any of the labels well knownas nucleic acid “markers.” Also, one could subject the transcripts toany of the well known amplification assays, such as PCR, LCR and soforth. Of course, one could also use an assay of the type describedsupra, wherein a reporter molecule is recombined into the cell, whereexpression of the reporter molecule is dependent upon the phosphorylatedexpression of one or more STAT molecules. Similarly, while full lengthreceptor molecules may be used, so can truncated forms of thesemolecules, as well as modified forms which present one or more aminoacid additions, deletions, or substitutions, but which nonethelessmaintain their ability to bind to IL-22 or an IL-22 fragment or variant.

[0049] In addition to, or instead of the molecules mediated by STATmolecules, one may measure phosphorylation levels of kinases, such asJAK-1 and/or Tyk-2, as well as the member of the MAPK family describedherein, including, but not being limited to, ERK1/2, MEK1/2, p90RSK,JNK/SAPK and/or p38, whose phosphorylation, activity and ability toinduce additional expression is upregulated by IL-22. Any combination ofthese molecules may be measured on the phosphorylation level.

[0050] In addition to, or as an alternative to, the DNA based assaysdescribed, supra, one may assay for the interaction described supra viaan immunoassay. As noted, the levels of phosphorylation of STAT1, 3, 5,JAK-1, Tyk-2, ERK1/2, MEK1/2, p90RSK, JNK/SAPK and/or p38 may bemeasured. One may use antibodies which bind specifically to one or moreof these molecules, but more preferably one uses antibodies whichspecifically bind to activated forms of these molecules such asphosphorylated forms. More specifically, one can use immunoassays whichtarget tyrosine phosphorylation of the target molecules, and in the caseof STAT3 for example, serine phosphorylation.

[0051] Any of the standard immunoassays will known to the art may beused, including western blotting, as described supra, radioimmunoassays,enzyme linked immunosorbent assays, precipitation assays, assaysinvolving solid phase/liquid phase separation, and so forth.

[0052] Similarly, i.e., STAT1, 3, 5, JAK-1, Tyk-2, ERK1/2, MEK1/2,p90RSK, JNK/SAPK and p38 all have specific well defined functions incells, one may measure a feature directly or indirectly influencedthereby, as a measure of the interaction.

[0053] In this way, any of the various methods can be used to determineagonists or antagonists of IL-22 or IL-22 receptors, such as antibodies,antibody fragments, IL-22 replacements, and so forth.

[0054] As was shown, supra, while the effects of IL-22 and IL-10 overlapsomewhat, there are differences. These differences may be expiated in,e.g., determining if an individual, a cell type, a population of cells,etc., is under or overexpressing IL-22 or IL-10. For example, it wasshown, supra, that IL-22 is involved in phosphorylation of serineresidues in STAT3, while IL-10 is not. Hence, by specificallydetermining a feature such as the level of serine phosphorylation inSTAT3, one can determine if IL-22 is being over an underexpressed.

[0055] Also a feature of the invention is a method for determining thelength of time after which a cell has been exposed to IL-22 or areceptor binding fragment, as it has been determined that while certainmolecules are phosphorylated immediately, but that the phosphorylatedforms decrease remarkable fast, others maintain phosphorylation forextended periods of time. Hence, if e.g., STAT1 and/or STAT3 are presentin phosphorylated form, then either stimulation with IL-22 is ongoing,or has taken place recently. In contrast, presence of highconcentrations of phosphorylated STAT3 need not indicate thatstimulation was recent.

[0056] The examples also indicate that JAK-1 is critical for efficacy ofIL-22. This suggests another embodiment of the invention, which is amethod to modify effect of IL-22 on a cell, either by adding JAK-1thereto or a portion of the JAK-1 molecule which interacts withcomplexes of IL-22 and an IL-22 receptor, or by adding a JAK-1 inhibitorwhich prevents the JAK-1 molecule from interacting in the mannerdescribed supra. This modulators of JAK-1 function may act on theprotein level, or may act on the transcription, such as promating orinhibiting phosphorylation. Similarly, mediators of the IL-22/IL-22Rbinding described herein may be used as therapeutic agents, to treatsubject's suffering from conditions characterized by inappropriateexpression of the molecules in the pathways described herein, includingthe STAT molecules, JAK-1, Tyk-2, MEK1/2, p90RSK, JNK/SAPK, and p38.These antagonists or agonists can be administered in manners well knownto the art and need not be reiterated here.

[0057] Other features of the invention will be clear to the skilledartisan and need not be reiterated further.

We claim:
 1. A method for identifying a substance which mediatesinteraction between interleukin-22 (IL-22) and an interleukin -22receptor (IL-22R), comprising admixing an IL-22 molecule or a receptorbinding fragment thereof, a cell which presents an IL-22 receptor or anIL-22 receptor binding fragment thereof on its surface and saidcompound, determining activation of a molecule that is expressed by saidcell, said molecule selected from the group consisting of STAT1, STAT3,STAT5, JAK-1, Tyk2, ERK1/2, MEK1/2, p90RSK, JNK/SAPK and p38, andcomparing activation of said molecule to activation when said cell whichpresents an IL-22R or IL-22 binding fragment thereof is admixed withIL-22 or an IL-22R binding fragment, any differences therebetweenindicating said substance mediates said binding.
 2. The method of claim1, comprising determining said activation via an immunoassay.
 3. Themethod of claim 1, comprising determining said activation via measuringnucleic acid expression of said molecule.
 4. The method of claim 2,wherein said immunoassay comprises an assay using an antibody whichbinds specifically to a phosphorylated form of said molecule.
 5. Themethod of claim 4, wherein said antibody binds specifically tophosphorylated serine on STAT3.
 6. The method of claim 1, comprisingdetermining said expression via a hybridization assay.
 7. The method ofclaim 3, comprising determining said activation by transfecting ortransforming said cell with a nucleic acid molecule which generates adetectable signal upon activation of said molecule.
 8. A method fortreating a subject suffering from a condition associated withinappropriate expression of a molecule selected from the groupconsisting of STAT1, STAT3, STAT5, JAK-1, Tyk2, ERK1/2, MEK1/2, p90RSK,JNK/SAPK and p38, comprising administering to said subject an amount ofa mediator of binding between IL-22 and an IL-22R sufficient toameliorate said inappropriate expression.
 9. A method for treating asubject suffering from a condition associated with inappropriateactivity of IL-22 comprising administering to said subject an inhibitorof JAK-1, Tyk2, ERK1, MEK1, p90RSK, JNK or p38 sufficient to inhibitIL-22 activity.